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| United States Patent |
6,200,753
|
|
Nathan
|
March 13, 2001
|
Detection of nucleic acid sequences
Abstract
A method for increasing the specificity of hybridization between a nucleic
acid probe and a nucleic acid sequence to be detected, by addition of
blocker molecules, which are complementary to the probe, raise of
temperature in order to melt non-perfectly matched hybrids of probe and
detected nucleic acid sequences, and lowering of the temperature again.
| Inventors:
|
Nathan; Asher (Bet-Shemesh, IL)
|
| Assignee:
|
Intelligene Ltd. (Jerusalem, IL)
|
| Appl. No.:
|
031532 |
| Filed:
|
February 27, 1998 |
Foreign Application Priority Data
| Jun 03, 1993[IL] | 105894 |
| Jun 02, 1994[WO] | PCT/US94/06034 |
| Current U.S. Class: |
435/6; 435/91.21; 536/24.3; 536/24.33 |
| Intern'l Class: |
C12Q 001/68 |
| Field of Search: |
435/6,91.21
536/23.1,24.3,24.1,24.33,24.5
|
References Cited
U.S. Patent Documents
| 5215899 | Jun., 1993 | Dattagupta | 435/6.
|
| 5348853 | Sep., 1994 | Wang et al. | 435/6.
|
| 5356774 | Oct., 1994 | Axelrod et al. | 435/6.
|
| 5369003 | Nov., 1994 | Reischl et al. | 435/6.
|
| 5434047 | Jul., 1995 | Arnold | 435/6.
|
| 5532126 | Jul., 1996 | Chu et al. | 435/6.
|
| 5814492 | Sep., 1998 | Carrino et al. | 435/91.
|
| 5952202 | Sep., 1999 | Aoyagi et al. | 435/91.
|
| Foreign Patent Documents |
| WO 89/05631 | Jun., 1989 | FR.
| |
| WO 89/05533 | Jun., 1989 | SE.
| |
Other References
Kohli, V. et al., Analytical Biochemistry, vol. 208, 1993; "Comparison of
in Vitro Transcriptions Using Various Types of DNA Templates", pp.
223-227.
Konarska, M.M. et al., Cell, vol. 63, 1990, "Structure of RNAs Replicated .
. . Polymerase", pp. 609-618.
Leary, S.L. et al., Gene, vol. 106, 1991, DNA-dependent RNA Polymerase . .
. Vitro, pp. 93-96.
Kwoh, D.Y. et al., Proceedings of the Nat. Academy of Sci. USA, vol. 86,
Feb. 1989, "Transcripton-based amplification system and detection . . .
format", pp. 1173-1177.
Chetverin, Alexander B. et al., On the Nature of Spontaneous RNA Synthesis
by QB Replicase, J. Mol. Biol. 1991, 222, p. 3-9.
Nath, Kamalendu et al., Covalent Attachment of Polyribonucleotides . . .
Ligase, J. Biol. Chem., vol. 219, No. 12, p. 3680-88, 1974.
Moore, Melissa J. et al., Site-Specific Modification of Pre-mRNA . . .
Sites, Science, vol. 256, 1992, p. 992-97.
Milligan, John F. et al., Oligonucleotide Synthesis Using . . . Templates,
Nucl. Acids Research, vol. 15, No. 21, 1987, p. 8783-8798.
Barany, Francis, Genetic Disease Detection . . . Ligase, Proc. Natl. Acad,
Sci. USA, vol. 88, p. 189-193, 1991.
Proceedings of the Nat. Academy of Sciences, USA , vol. 87, issued Mar.
1990, J.C. Guatelli et al, "Isothermal, in vitro, amplification of nucleic
acids by a multienzyme reaction modeled after retroviral replication", pp.
1874-1878, see entire document.
|
Primary Examiner: Elliott; George C.
Assistant Examiner: Schmidt; Melissa
Attorney, Agent or Firm: Blank Rome Comisky & McCauley LLP
Parent Case Text
This application is a continuation in-part of U.S. application Ser. No.
08/556,940, filed Apr. 9, 1996, now U.S. Pat. No. 5,871,914, which was the
National Stage of International Application No. PCT/US94/06034, filed May
2, 1994.
Claims
What is claimed is:
1. A method for eliminating hybridization of a non-perfectly matched
nucleic acid sequence, which is contained in an assayed sample, to another
nucleic acid sequence, forming a part of a detection ensemble, the method
comprising the steps of:
(a) incubating a reaction mixture comprising an assayed nucleic acid
sequence which is presented in said assayed sample and a detection
ensemble under conditions allowing hybridization of a perfectly matched
nucleic acid sequence;
(b) increasing a temperature of the reaction mixture to the temperature
which is below the melting point of said perfectly matched hybridized
nucleic acid sequence, but being sufficiently high to cause melting of
said non-perfectly matched nucleic acid sequence; and
(c) adding an amount of a blocker nucleic acid sequence, having a sequence
which perfectly matches said another nucleic acid sequence, being part of
the detection ensemble, the blocker nucleic acid sequence being
sufficiently long to block hybridization of said non-perfectly matched
nucleic acid sequence contained in said assayed sample to said another
nucleic acid sequence upon lowering of said temperature;
whereby said blocker nucleic acid sequence hybridizes to said another
nucleic acid sequence, forming part of said detection ensemble, which was
previously melted in step (b) and eliminating the hybridization of the
non-perfectly matched nucleic acid sequence to said another nucleic acid
sequence.
2. The method according to claim 1, wherein the amount of blocker nucleic
acid sequence added in step (c) is greater than the amount of said another
nucleic acid sequence.
3. The method according to claim 1, wherein said another nucleic acid
sequence has an arbitrary sequence not presented in said assayed nucleic
acid sequence contained in said assayed sample.
4. A kit for performing a hybridization assay, in which hybridization of
non-perfectly matched nucleic acid sequences, which are contained in an
assayed sample, to another nucleic acid sequence, which is contained in a
detection ensemble, is eliminated, the kit comprising:
(i) a detection ensemble comprising said another nucleic acid sequence to
be contained in the assayed sample in a first container, and
(ii) blocker nucleic acid sequences which are perfectly matched to said
another nucleic acid sequences to be contained in a second container.
5. A kit according to claim 4, further comprising reagents necessary for
hybridization of nucleic acid sequences.
6. A method for avoiding hybridization of a non-perfectly matched nucleic
acid sequence present in a sample to a probe nucleic acid sequence, the
method comprising the steps of:
(a) incubating a reaction mixture of the sample having an assayed nucleic
acid sequence and the probe nucleic acid sequence under conditions
allowing hybridization of a perfectly matched nucleic acid sequences;
(b) increasing a temperature of the reaction mixture to the temperature
which is below the melting point of said perfectly matched hybridized
nucleic acid sequences but above that which leads to melting of said
non-perfectly matched hybridized nucleic acid sequences; and
(c) adding an amount of a blocker nucleic acid sequence, having a sequence
which perfectly matches said probe nucleic acid sequence, the blocker
nucleic acid sequence being sufficiently long to block hybridization of
said non-perfectly matched nucleic acid sequence contained in said sample
to said probe nucleic acid sequence upon lowering of said temperature;
said blocker nucleic acid sequence hybridizes to said probe nucleic acid
sequence, which was previously melted in step (b) eliminating thereby
hybridization of said non-perfectly matched nucleic acid sequence, to said
probe nucleic acid sequence.
7. The method according to claim 6, wherein the amount of blocker nucleic
acid added in step (c) is higher than the amount of said probe sequence.
8. The method according to claim 6, wherein said probe nucleic acid
sequence has a sequence not presented in said assayed nucleic acid
sequence contained in said assayed sample.
9. A kit for performing a hybridization assay, while avoiding hybridization
of non-perfectly matched nucleic acid sequences, presented in an assayed
sample to a probe nucleic acid sequence, the kit comprising:
(i) probe nucleic acid sequences provided in a first container; and
(ii) blocker nucleic acid sequences which are perfectly matched to said
probe nucleic acid sequences provided in a second container.
10. The kit according to claim 9, further comprising reagents necessary for
hybridization of nucleic acid sequences.
Description
FIELD OF THE INVENTION
The present invention concerns a method and kit for the detection of
specific nucleic acid sequence in a sample.
BACKGROUND OF THE INVENTION
Detection of the presence of a specific DNA or RNA sequence in a sample is
required for a variety of experimental, diagnostic and therapeutic
purposes, e.g. detection of a specific mutation in a sample of amniotic
fluid, parenterage testing, testing for incorporation of a viral DNA into
a cell's genomic DNA, etc. The task of direct detection of a specific DNA
or RNA sequence, which is routinely performed by the use of an
appropriately labelled probe, is often hindered by the fact that the
specific DNA or RNA is present in a sample only in minute amounts.
Examples of methods which enable the amplification of DNA sequences present
in a sample in only minute quantities are: LCR (ligase chain reaction),
3SR (self-sustained sequence replication) or PCR (polymerase
chain-reaction). In PCR a sample is contacted with a primer DNA
complimentary to a 3' end sequence of the specific DNA, a DNA polymerase
and with single DNA nucleotides. Following a number of replication cycles,
the sample is enriched with the specific assayed DNA. A typical cycle of
PCR comprises three distinct stages: a first stage in which the
double-stranded DNA is melted to two single strands; a second stage of
annealing of the primer to the single-stranded DNA; and a third stage of
polymerization where the annealed primers are extended by the DNA
polymerase, to produce a double-stranded DNA. The cycle of melting,
annealing and DNA synthesis is repeated many times, the products of one
cycle serving as templates for the next ad thus, each successive cycle
enriches the sample with the specific DNA.
PCR suffers from several shortcomings, the most serious of which being its
lack of specificity. The effective hybridization temperature, i.e. the
temperature in which the two strands of DNA hybridize, determines the
specificity of the reaction. A low effective hybridization temperature
results in a higher percentage of non-specific binding. In PCR this
temperature, which is defined by the temperature of the annealing stage,
is relatively low and this brings about non-specific binding of the probe
to the target sequences resulting in amplification of undesired sequences
which brings about a relatively high background reading.
This non-specificity also requires an additional and time-consuming
detection procedure such as electrophoretic separation of the
amplification products on an agarose gel, in order to separate between the
various amplification products, and does not enable detection of the
presence of the assayed DNA by a mere detection of amplification.
PCR also suffers from a severe problem of contamination which is due to
amplification of sequences that did not originate from the test sample
being sequences unintentionally introduced to the sample.
Another disadvantage of PCR is that it is a complex procedure. Typically,
each of the stages of melting, annealing and polymerization is carried out
at a different temperature, e.g. melting at 94.degree. C., annealing at
50.degree. C. and polymerization at 72.degree. C. Since the samples have
to be constantly cycled through several temperatures a special apparatus
is required rendering the procedure laborious and time consuming.
Another shortcoming of PCR is in the time required therefor. A typical
cycle lasts several minutes, and usually 25-30 cycles are required to
produce sufficient copies of amplified DNA. Thus, a typical PCR even in a
completely automated system lasts at least 2 to 3 hours.
Finally, PCR is basically suited for the detection of DNA sequences. Where
detection of RNA sequences is desired, RNA has to be converted first to
DNA (by reverse transcription). This conversion to DNA requires additional
time, effort and enzymes, and also introduces many errors due to the
inherent inaccuracy of reverse transcription.
It should be noted that although PCR is advantageous in obtaining large
amounts of a specific DNA, such as for producing large quantities of
probes for genetic assays, it is often an "over-kill" where merely the
presence of a specific DNA sequence in a sample is to be assayed.
Other such methods such as 3SR (WO PCT 89/05631) and Target Nucleic Acid
Amplification/Detection (WO PCT 89/05533) are relatively rapid isothermal
processes for DNA detection. However, these methods also suffer from
relatively effective low hybridization temperatures which are even lower
than those of PCR, typically in the range of 37-41.degree. C. These low
temperatures drastically reduce the specificity of the procedure due to
non-specific probe-target binding, and in cases of clinical diagnostics,
this may result in an intolerable level of misdiagnosis.
Additionally, amplification strategies such as Target Nucleic Acid
Amplification/Detection that are based on the amplification properties of
a replicase-type enzyme are unreliable due to the possibility of
spontaneous RNA amplification in the absence of target (Chetverin-AB, et
al., J. Mol. Biol., 222(1), 3-9 (1991)).
It is the object of the invention to provide a method for the detection of
a nucleic acid sequence which is:
(i) reliable and sequence specific due to the minimalization of incorrect
target-probe hybridization;
(ii) relatively rapid;
(iii) essentially isothermic eliminating the need for specialized and
expensive apparatus;
(iv) relatively simple, not requiring the addition of a large number of
different enzymes or nucleotide pools; and
(v) amenable to automation by enabling the amplification process itself to
be indicative of the presence or absence of the nucleic acid sequence to
be assayed.
U.S. Pat. No. 5,434,047 teaches a method for ensuring that only hybrids
which are perfectly matched between a probe sequence (termed "target
probe") and a nucleic acid sequence present in a sample (termed "target
nucleotide sequence") are formed, while imperfect matches between the
probe and other sequences present in the assayed sample (termed
"non-target nucleotides") are not formed. The method involves adding to
the reaction mixture blocker molecules which are complementary to the
non-target nucleotides which are present in the assayed sample. These
blocker molecules, hybridize with the non-target nucleotide in the assayed
sample, avoiding their hybridization with the target probe, and thus
eliminate production of false-positive results. Each blocker molecule, of
U.S. Pat. No. 5,434,047, is specific only to one type of non-target
nucleotide, and is emphatically not universal in all assay kits. For
example, where it is desired to assay a sample for the presence of a
specific nucleic acid sequence ("target nucleotide") which is indicative
of a specific bacteria species, a battery of different blocker molecules,
each complementary to a nucleic acid sequence of other species of bacteria
("non-target nucleotides") have to be constructed. If, for some reason,
not all possible non-target nucleotide combinations were predicted, and
consequently not all types of complementary blocker molecules were
constructed, the blocker molecule would not avoid imperfect matches with
the labeled probe, thus providing a false-positive result.
It would have been desirable to construct a universal single blocker
molecule, which would be suitable for elimination of all imperfect
hybridizations between a probe and nucleic acid sequences present in a
sample, and thus eliminate all positive results, even in the presence of
many types of non-target nucleotide sequences.
Further objects of the invention will become clear from the following
description.
Glossary
Below are the meanings of some of the terms which will be used in the
following description and claims. For ease of reference, the reader is
also referred to the accompanying drawings (the numbers in brackets in the
Glossary below refer to the item numbers in the drawings):
Assayed nucleic acid sequence (102,202,302,402,502,602,1402)--The DNA or
RNA sequence which presence in the sample is to be detected.
First DNA molecule (220,320,420,520,620,1420)--a DNA molecule having a
double-stranded, i.e. functional promoter and a 5' end sequence which is
complementary to the 5' end portion of the assayed nucleic acid sequence
(102, . . . etc.).
Second DNA molecule (222,322,432,522,622,1422)--a DNA molecule comprising a
single-stranded 3' end sequence being complementary to the 3' end portion
of the assayed nucleic acid sequence (102, . . . etc.) and further
comprising a sequence which can be transcribed to the triggering RNA
sequence (see below). The 3' end sequence of the second DNA molecule and
the 5' end sequence of the first DNA molecule may be complementary to the
entire assayed nucleic acid sequence or to only a part thereof, leaving an
intermediary portion in the assayed nucleic acid sequence having no
complementary counterparts in either the first or second DNA molecules.
Third DNA molecule (623)--a single-stranded DNA molecule complementary to
the intermediary portion of the assayed nucleic acid sequence.
Detection ensemble (104,204)--an ensemble of molecules comprising the first
DNA molecule (220, . . . etc.), the second DNA molecule (222, . . . etc.),
and where the 5' end sequence and the 3' end sequence of the first and
second DNA molecule, respectively, are complementary together to only a
portion of the assayed nucleic acid sequence also comprising the third DNA
molecule (623). The detection ensemble optionally comprises also a ligase.
In the presence of the assayed DNA (102, . . . etc.) and the transcription
reagents (see below) the detection ensemble is activated and an RNA
transcript
(110,210,310,410,510,610,710,810,910,1010,1110,1210,1310,1410,1510, 1610)
comprising the triggering RNA sequence (see below) is produced.
Triggering RNA sequence--a sequence in the RNA transcript (110, . . . etc.)
transcribed from the second DNA molecule (222, . . . et.), which is only
produced after activation of the detection ensemble. This RNA sequence is
then capable of triggering transcription in the RNA amplification ensemble
(see below) of a signal RNA molecule (see below) comprising the signal RNA
sequence (see below).
Triggering RNA molecule (110,210,310,410,510,610,710,810,910,1010,
1110,1210,1310,1410,1510,1610)--the RNA molecule comprising the triggering
RNA sequence.
Signal RNA sequence--a sequence in the transcription product of the RNA
amplification ensemble (see below). The production of the signal RNA
sequence, above a baseline level, indicates the presence of the assayed
nucleic acid sequence in the sample.
Signal RNA molecule (116,716,816,916,1016,1116,1216)--the RNA molecule
comprising the signal RNA sequence.
Signal DNA sequence--a DNA sequence serving as a template from which the
signal RNA sequence is transcribed.
Transcription reagents (113,213,413,513,713,813,913,1013,1113,1213,
1313,1413,1513,1613)--RNA polymerase with single RNA nucleotides and
buffers required for RNA transcription.
Transcription system--a DNA homoduplex or a DNA/RNA heteroduplex comprising
a functional promoter and a downstream DNA or RNA sequence which can be
transcribed upon activation of the promoter into an RNA transcript.
RNA amplification ensemble (FIGS. 7-13, 15,16)--an ensemble comprising
essentially the triggering RNA molecule (110, . . . etc.) and the
transcription reagents (213, . . . etc.) in the first embodiment of the
invention; or the triggering RNA molecule (116, . . . etc.), the
transcription reagents (213, . . . etc.) and fourth DNA molecule (see
below) in the second embodiment; or the triggering RNA, the transcription
reagents and a fifth and sixth DNA molecules (see below), in the third
embodiment of the invention; or the triggering RNA, the transcription
reagents, and a seventh and eighth DNA molecule in the fourth embodiment
of the invention. The RNA amplification ensemble optionally comprises a
ligase. In one embodiment of the first embodiment, the presence of the
triggering RNA together with the transcription system is sufficient for
the transcription of the signal RNA sequence. In the second embodiment,
the triggering RNA hybridizes with the fourth DNA molecule. In the third
embodiment, the triggering RNA hybridizes with the fifth and sixth DNA
molecules bringing them together. In the fourth embodiment the triggering
RNA hybridizes with the seventh or eighth DNA molecules. The RNA/DNA
hybrid produced in accordance with the second, third and fourth
embodiments, serves as a template for the production of the signal RNA
sequence.
Fourth DNA molecule--a DNA molecule (1148) which is part of the RNA
amplification ensemble in accordance with the second embodiment. This
molecule comprises a promoter which is single-stranded in at least an
essential part thereof and is thus inactive. It further comprises the
signal DNA sequence. When the triggering RNA sequence, which in this
embodiment is complementary to the single-stranded part of the promoter,
hybridizes with the single-stranded part of the promoter of the fourth DNA
molecule, a functional promoter is produced and thus the signal RNA
molecule can be transcribed.
Fifth DNA molecule--a molecule (1252) which is part of the amplification
ensemble in accordance with the third embodiment. It comprises a
functional promoter, and at its 5' end, a single-stranded sequence which
is complementary to the 5' end portion of the triggering RNA sequence.
Sixth DNA molecule--a molecule (1259) which is part of the amplification
ensemble in accordance with the third embodiment. It comprises at its 3'
end a single-stranded sequence which is complementary to the remaining 3'
end portion of the triggering RNA sequence and in addition, comprises the
signal DNA sequence.
Seventh DNA molecule--a molecule (1580) which is part of the amplification
ensemble in accordance with the fourth embodiment. It has a functional,
double-stranded promoter, either a priori prepared or assembled from two
single stranded sequences, linked to an antisense sequence complementary
to the 3' end sequence of the triggering RNA. One or a few end nucleotides
in the 5' end of the template strand of this molecule could be RNA
nucleotides. The 3' end sequence of the triggering RNA hybridizes to said
antisense sequence and after ligation the promoter can induce RNA
transcription, the triggering RNA serving as a template.
Eighth DNA molecule--a molecule (1529') which is part of the amplification
ensemble in accordance with the fourth embodiment. It is similar to the
seventh DNA molecule, the difference being in the antisense sequence which
in the eighth DNA molecule is identical to the 5' end sequence of the
triggering RNA molecule. The transcription product of the seventh DNA
molecule/triggering RNA hybrid can thus hybridize to the eighth molecule
and the so formed hybrid serves there as a template for transcription of
RNA molecule having the sequence of the triggering RNA sequence, which in
turn can activate again the seventh DNA molecule in a "ping-pong" manner.
Promoter molecule--a DNA molecule (1429) which is part of the detection
ensemble according to the fourth embodiment of the invention and is
essentially identical to the eighth DNA molecule.
Adapter molecule--a DNA molecule (1431) comprising a sequence complementary
to the non-template sequence immediately adjacent the promoter sequence in
the promoter molecule possibly having one or a few RNA nucleotides at its
3' end.
Joiner molecule--a DNA molecule (1433) comprising at its 5' end a sequence
which is complementary to a 5' end portion of the adapter molecule and a
sequence in the remaining 3' portion of the molecule which is
complementary to 3 end portion of the first molecule in accordance with
the fourth embodiment. The joiner molecule serves for joining the first
and the adapter molecule in the fourth embodiment.
DNA iniation sequence (DIS)--a DNA sequence present downstream of the
promoter of the seventh or eighth DNA molecules which enhances
transcription of the sequence present downstream therefrom, by the RNA
polymerase.
Probe nucleic acid sequence--a nucleic acid sequence which is complementary
to a pre-defined nucleic acid sequence whose presence is to be detected in
the assayed sample.
Blocker nucleic acid sequence--a nucleic acid sequence which is
complementary to the probe nucleic acid sequence.
Perfectly matched--two nucleic acid sequences which are fully complementary
to one another.
Non-perfectly matched--two nucleic acid sequences which are not fully
complementary to one another.
SUMMARY OF THE INVENTION
The present invention is based on a novel concept for the detection of a
nucleic acid sequence present in a sample (assayed nucleic acid). The
present invention is useful for the detection of a nucleic acid sequence
which may be DNA or RNA having a known sequence. The method is based on a
system comprising two components: a detection ensemble and an RNA
amplification ensemble.
If the assayed nucleic acid sequence is present in the sample, the
detection ensemble gives rise to the production of a triggering RNA
sequence. The triggering RNA sequence can initiate an RNA amplification
reaction in the RNA amplification ensemble, to produce a signal RNA
sequence, which can then be detected by means known per se. If the assayed
DNA sequence is not present in the sample, the triggering RNA sequence and
consequently the signal RNA sequence are not produced. Thus, the presence
or absence of the signal RNA sequence indicates the presence or absence,
respectively, of the assayed DNA sequence in the sample.
As can be seen, the presence of the assayed sequence is only required for
the first step, in the initial detection ensemble and is no longer
required for the RNA amplification ensemble. This uncoupling of the
detection and amplification steps allows for various manipulations of the
detection ensemble such as addition of blocker molecules and the raising
of temperatures, which reduces non-specific hybridization. For example,
after hybridization, the temperature may be raised once to a temperature
in which all non-specific hybrids will melt and only the specific hybrid
will remain. All said manipulations have to be carried out only once,
during the detection, and the cycles of amplidnot require any intervention
and can thus be easily automized.
Furthermore, the detection of the presence of the assayed nucleic acid
sequence results in RNA amplification which may for example, be detected
by light absorbance at a frequency of 260 nm, with no need to open the
reaction vessel and separate the various amplification products. The
detection of RNA amplification per se obviating the need to determine
whether a specific amplification product is present in the reaction
mixture considerably simplifies the whole process as well as eliminates
contamination of other reaction vessels by aerosol particles from the
assayed reaction vessel which is a current problem encountered in other
amplification procedures.
In accordance with the present invention, there is no need to amplify the
assayed DNA sequence, but rather its presence brings about production of
large quantities of the signal RNA sequence. The method of the invention
thus involves production and amplification of RNA rather than
amplification of DNA as in PCR. Consequently there is no need for melting
of the two DNA strands during amplification cycles since the RNA removal
from its template occurs during the normal course of transcription, and
accordingly the repetitive temperature cycles of PCR are avoided.
Another advantage of the method of the invention is that, contrary to DNA
replication, in RNA transcription several RNA polymerase enzymes can
operate on the same template simultaneously and thus the overall
transcription process is relatively rapid. Furthermore, the speed of RNA
production can be increased if the RNA molecule produced in the
amplification ensemble and which comprises the signal RNA sequence,
comprises also the triggering RNA sequence itself, which triggering
sequence can in turn activate additional transcription in a
self-amplifying manner, and thereby the production of RNA advances
exponentially in a very rapid manner.
Thus, the method of the invention provides a relatively specific, rapid and
uncomplicated method for the detection of an assayed nucleic acid sequence
in a sample.
The present invention provides a method for detecting the presence of an
assayed nucleic acid sequence in a sample, comprising the steps of:
(a) reacting the sample with a detection ensemble comprising:
a first DNA molecule having a promoter sequence and a 5' end sequence which
is complementary to the 5' end portion of the assayed nucleic acid
sequence;
a second DNA molecule comprising a single-stranded 3' end sequence being
complementary to a 3' end portion of the assayed nucleic acid sequence,
and further comprising a sequence which can be transcribed into a
triggering RNA sequence capable of initiating a reaction in an appropriate
transcription system in which an RNA molecule having a signal RNA sequence
is being produced; the 3' end sequence of the second DNA molecule and the
5' end sequence of the first DNA molecule may be complementary to the
entire assayed nucleic acid sequence or to only a part thereof leaving an
intermediary portion in the assayed nucleic acid having no complementary
counterpart in either the first or the second DNA molecules, in which case
the detection ensemble further comprises
a third DNA sequence being complementary to said intermediate portion;
(b) incubating under conditions to allow hybridization of said first DNA
molecule and said second DNA molecule and were present also said third DNA
molecule with said assayed nucleic acid sequence, and optionally adding a
ligase to allow ligation of adjacent ends of said first, second and third
DNA molecules;
(c) adding transcription reagents comprising an RNA polymerase and RNA
nucleotides and incubating under conditions to allow the formation of RNA
transcripts having said triggering RNA sequence;
(d) contacting the RNA transcripts with an RNA amplification ensemble in
which the triggering RNA sequence induces formation of RNA molecules
containing the signal RNA sequence; and
(e) detecting the presence of said signal RNA sequence, positive results
indicating the presence of said assayed nucleic acid sequence in said
sample.
Said first molecule may comprise a double-stranded and hence functional
promoter. Alternatively, the promoter is a priori single-stranded in at
least an essential part thereof and a sequence complementary to the
single-stranded portion of the promoter is added during or after step (b).
Namely, it should be understood that by the above definition of first DNA
molecule, the functional promoter may be present a priori or may be
assembled in situ during the performance of the assay in the assay vessel.
Steps (a) and (b) of the method of the invention may be modified to
increase the specificity of the detection and/or prevent production of
short sequences of RNA transcribed from the first DNA molecule, which may
increase the background signal. These modifications include, for example,
an additional step after step (b) of raising the temperature to a point
where only perfectly matched hybrids of assayed nucleic acid sequences and
first and second DNA molecules (and also third DNA molecule if present)
remain hybridized while all other hybrids in which the individual strands
do not perfectly match one another are melted. The reformation of
mismatched hybrids after melting can be prevented by the addition of
blocker molecules which compete with the assayed nucleic sequence by
hybridizing at a high affinity to the first or to the second DNA
molecules. Such a modification ensures that triggering RNA is produced
only in case of a perfect match between the assayed nucleic acid sequence
and the first and second DNA molecules.
In order to avoid production of undesired short RNA transcripts from the
first DNA molecule which would have an effect of increasing assay "noise",
it is possible to assemble the promoter of the first DNA molecules in
stages. In this case, the first molecule comprises a promoter which is
single-stranded in at least an essential part thereof and thus
non-functional. After the formation of hybrids of the assayed nucleic acid
sequence and the first and second DNA molecules, blocker DNA or RNA
molecules are added which hybridize only to the free first DNA molecules
in such a manner so as to avoid subsequent hybridization thereto of the
missing promoter part and cannot hybridize to first DNA molecules present
in the hybrid. A DNA molecule comprising the missing promoter part is then
added, which completes only the promoter of first DNA molecule in said
hybrid rendering it functional and thus enabling the production of the
triggering RNA. In contrast to this, the molecule comprising the missing
promoter is unable to hybridize with free first DNA molecules which are
blocked, and thus no short RNA transcripts are produced from free first
DNA molecules.
Free first DNA molecules can also be separated from hybrids of assayed
nucleotides and first and second DNA molecules, for example, by having the
second DNA molecules bound to a solid support, e.g., magnetic beads and
thus, after hybridization, removing all non-bound, i.e. free, DNA
molecules.
If the production of short RNA transcripts from the sample is avoided, it
is possible to detect the presence of the assayed nucleic acid sequence by
detecting the mere amplification of RNA per se, for example, by a change
in the absorbance, with no need for the detection of the presence of a
specific signal sequence.
Finally, it is possible to determine whether the only transcript produced
is a short transcript transcribed from the first DNA molecule or whether
in addition, triggering RNA is produced by using synthetic, nucleotides
labeled with a fluorescent moiety. One type of these non-naturally
occurring nucleotides which are recognized by the RNA polymerase, may be
introduced in the coding region first DNA molecule, and a second type may
be introduced in the second DNA molecule. The synthetic nucleotides
complementary to those present in the first DNA molecule are labeled with
one type of fluorescent, i.e. yellow. The synthetic nucleotides
complementary these present in the second DNA molecule are labeled by
another type of fluorescent, i.e. blue. After the reaction takes place,
the free synthetic nucleotides are separated from the reaction mixture,
for example by washing through a charged filter through which the neutral
synthetic nucleotides pass. If only short RNA transcripts from the first
DNA molecule are produced the fluorescence in the reaction mixture will be
totally yellow. If triggering RNA transcribed from both the first and
second RNA molecules is produced the fluorescence will be both yellow and
blue and will be seen as a green color.
In accordance with a first embodiment of the present invention, the
triggering RNA sequence comprises a sequence which can bring about,
directly or by hybridizing to various DNA molecules, the production of
self-replicating RNA, namely an RNA which serves as a template for
formation of identical RNA molecules by an RNA polymerase. Examples of
such self-replicating RNA's are X-RNA and Y-RNA (Konarska, M M., Sher, P.
A., i Cell, 63(3), 608-18, (1990). In accordance with the first
embodiment, the RNA amplification ensemble, comprises RNA nucleotides and
an RNA polymerase and at times DNA molecules which are required in some
embodiments for assembling a functional promoter. The signal RNA which is
produced in the RNA amplification ensemble is a self-replicating RNA. The
presence of self-replicating RNA in the sample, in accordance with the
first embodiment, indicates the presence of the assayed DNA sequence in
the sample.
In accordance with a second embodiment of the present invention, the
amplification ensemble comprises a fourth DNA molecule, which comprises a
promoter which is single-stranded in at least an essential part thereof,
and is thus inactive, and further comprises a signal DNA sequence which is
transcribed into the signal RNA sequence. In accordance with this
embodiment, the triggering RNA sequence is complementary to the essential,
single-stranded portion of the promoter. When the triggering RNA sequence
is contacted with the fourth DNA molecule, the two molecules hybridize
whereby the promoter becomes double-stranded and thus functional.
Consequently, in the presence of a transcription system, i.e. RNA
polymerase and RNA nucleotides, an RNA molecule including the signal RNA
sequence is transcribed. Preferably, the RNA molecule thus produced which
includes the signal RNA sequence includes also the triggering RNA sequence
and consequently the process is self-amplifying, namely, the RNA
transcripts produced themselves induce by themselves transcription of
additional RNA transcripts.
In accordance with a third embodiment of the invention, the amplification
ensemble comprises a fifth DNA molecule which has a functional promoter
and a single-stranded sequence at its 5' end and comprises a sixth DNA
molecule which has a 3' single-stranded end sequence. The triggering RNA
sequence is complementary to the above-mentioned two single-stranded
sequences, its 5' end sequence is complementary to the 5' end sequence of
the fifth DNA molecule and its remaining 3' end sequence is complementary
to the 3' end sequence of the sixth DNA molecule. Consequently, the
triggering RNA sequence after hybridizing to the single-stranded sequence
of the fifth DNA molecule and the sixth DNA molecule, brings the
respective 5' and 3' ends of the fifth and sixth DNA molecules together
whereupon they can be ligated by the use of the ligase. In the presence of
an RNA transcription system an RNA transcript comprising a signal RNA
sequence is produced. Similarly as in the second embodiment, the fifth DNA
molecule optionally comprises a sequence which transcribes into the
triggering RNA sequence and the RNA transcript produced therefrom serves
also as a trigger for production of additional such transcripts.
By a modification of the third embodiment, the sixth DNA molecule serves as
a template for transcription of a self-replicating RNA, such as the X-RNA.
Once a self-replicating RNA is produced, it serves as a template for
production of further self-replicating RNA molecules.
In accordance with a fourth embodiment of the invention, the amplification
ensemble comprises a seventh and an eighth DNA molecule both of which have
a functional, double-stranded promoter. The promoters may be, a priori
double-stranded or may be assembled in steps from a single-stranded
promoter. The seventh DNA molecule has an antisense sequence attached to
the non template strand of the promoter which is complementary to the 3'
end sequence of the triggering RNA. The eighth DNA molecule has an
antisense sequence attached to the promoter which is identical to the 5'
end sequence of the triggering RNA. When the triggering RNA is contacted
with the amplification ensemble in accordance with the fourth embodiment,
the RNA hybridizes to the short antisense sequence in the seventh DNA
molecule and after ligation the functional promoter can induce production
of RNA, wherein the triggering RNA can serve as template. The RNA
transcript thus produced can in turn hybridize in a similar manner to the
eighth DNA molecule and the RNA transcript which is produced there is
identical to the triggering RNA and can again activate the seventh DNA
molecule. Consequently there is a continuous cross-triggering of RNA
transcription and a large number of copies of both the triggering RNA and
the antisense RNA thereto are produced, which can both serve as the signal
RNA.
If desired, it is possible to construct a promoter made of a single DNA
strand which is able to loop and form a double-stranded part only under
proper conditions. When the promoter is not ligated to the RNA transcript,
the loop and hence the double-stranded part is not formed. Only when the
promoter is ligated to the RNA transcript the loop structure is stabilized
and a double-stranded, functional promoter is formed.
In accordance with a slight modification of the fourth embodiment, it has
been found that it is preferable to insert after the promoter a short DNA
initiation sequence (DIS) which is recognized at a high affinity by the
RNA polymerase and this increases considerably the transcription rate of
the RNA molecule (Milligan et al., NAR, 15, pp. 8783 (1987)). One variant
of DIS termed "DIS1" should be inserted after the promoter of the seventh
molecule and another variant of DIS termed "DIS2" should be added after
the promoter of the eighth molecule. Since the DIS is added after the
promoter, it becomes transcribed to the RNA molecule in each cycle of
amplification so that the transcribed RNA molecules becomes gradually
enriched with DIS1 or DIS2 on both ends and then the amplification process
is stopped after two cycles. In order to avoid this lengthening phenomena,
a ribozyme, which is an RNA sequence featuring catalytic properties and
capable of recognizing and cutting a specific RNA sequences, is introduced
to the reaction mixture. One type of ribozyme specifically cuts out the
RNA sequences transcribed from DIS1 and another ribozyme specifically cuts
out DIS2 after each amplification cycle. The sequence transcribed from
either DIS, termed hereinafter "dis1" and "dis2" may be cut immediately
after transcription when they are on the 5' end, or before hybridization
of the triggering RNA to its cognate promoter when it is on the 3'. The
latter option has some advantages. First higher fidelity at the 3' end.
Since the 3' fidelity of the polymerase is not high, the cutting off the
3' end of the transcript eliminates this problem. Another advantage
resides in the fact that cutting by the ribozyme becomes a prerequisite
for ligation which ensures the correct sequence of events.
In addition to the advantages stated above, the use of ribozymes in general
offers several advantages: first it enables higher specificity. The
recognition sequence of the ribozyme can be made highly specific so that
after each amplification cycle the specificity of the RNA molecule is
verified, thereby reducing background noise due to contamination of the
reaction by undesired nucleic acid molecules.
Second, ribozymes can replace need for a ligase. Some are to specifically
ligate two RNA molecules together, so that the nick that occurs after the
triggering transcript anneals to its promoter can be repaired by the
ribozyme instead of enzymatically.
Third, improved read-out strategies. There is a wide repertoire of ribozyme
enzymatic activities. Some ribozymes are able to add a single nucleotide
to the RNA sequence and some are able to cut a single nucleotide
therefrom, for example, during the ligation procedure. By using labeled
nucleotides these properties of the ribozymes can be used to ease
detection of the transcription products.
The present invention also provides a kit for carrying out the method of
the invention. The kit typically comprises the various DNA molecules,
reagent systems, etc. required for carrying it out. Separate kits for each
of the above-mentioned embodiments are provided.
By another aspect, the present invention concerns a method for avoiding
hybridization of a non-perfectly matched nucleic acid sequence present in
a sample to a probe nucleic acid sequence, the method comprising the steps
of:
(a) incubating the sample and the probe nucleic acid sequence under
conditions allowing hybridization of matched nucleic acid sequences;
(b) increasing the temperature of the reaction mixture to such which is
below the melting point of perfectly matched hybridized nucleic acid
sequences but above that which leads to melting of non-perfectly matched
hybridized nucleic acid sequences;
(c) adding an amount of a blocker nucleic acid sequence, having a sequence
which perfectly matches the sequence of said probe nucleic acid sequence
or matches an essential part of said probe, the blocking sequence being
sufficiently long to block hybridization of the nucleic acid sequence
contained in the sample to probe nucleic acid sequence upon lowering of
temperature;
whereby the blocker sequence hybridizes to said probe nucleic acid sequence
which was melted in step (b) eliminating hybridization of a non-perfectly
matched nucleic acid sequence, which is contained in the assayed sample,
to the probe
The method for avoiding hybridization of nucleic acids contained in a
sample, which are not perfectly matched to probe nucleic acid sequences,
is intended for increasing the specificity of hybridization of probes to
the sequence in a sample to ensure correct detection or correct
amplification of nucleic acid sequences. The method of the invention in
accordance with said another aspect minimizes "false positive results" due
to hybridization of non-perfectly matched sequences, present in a sample
to probe sequences (for example labeled probe sequences).
As a first step the probe sequences are allowed to hybridize with single
stranded nucleic acid sequences present in the assayed sample. Two types
of hybrids are formed:
(1) hybrids between probes and sequences in the assayed sample which are
perfectly matched;
(2) hybrids between probes and sequences in the assayed sample which are
non-perfectly matched.
As a second step, the temperature is raised to a temperature wherein
essentially all hybrids (2) above are melted, while at least some of
hybrids (1) are maintained. As a result, all probes which were hybridized
to non-perfectly matched sequences in the assayed sample are released.
As a third step, blocker molecules, which are complementary to the probe
sequence or a part thereof are added. These blocker molecules can
hybridize to the probe molecules which were melted in the second step, so
that when the temperature is lowered again, nucleic acids which were
present in the assayed sample and melted in the second step (due to
imperfect hybridization to the probe) cannot find any free probe sequences
and remain unhybridized.
At the end of the method, only the nucleic acid sequences, contained in a
sample, which were perfectly matched to the probe are hybridized thereto
(and can be detected, amplified etc.) and those nucleic acids which were
not perfectly matched, remain unhybridized.
The method of said another aspect is suitable as a step in many techniques
where it is important that only perfectly matched hybrids are formed, such
as in detection assays (where a perfect hybrid with a labeled probe is
required), in amplification techniques (where perfect hybrid with an
amplification primer is required), and in techniques involving nucleic
acid catalytic activity (where perfect hybrids with catalytic nucleic acid
sequences, i.e. ribozymes, is required) and the like.
In order to ensure preferred hybridization of the probe, released in the
second step, to the blocker added in the third step, an excess of blocker
over the probe and over nucleic acid sequence present in the sample should
be used.
In addition, or alternatively in order to ensure that the affinity of the
probe sequence to the blocker sequence is higher than the affinity of the
probe sequence to the sequence present in the sample, it is possible to
construct probes containing an arbitrary sequence which is not present in
the nucleic acid sequences in the sample.
A complementary arbitrary sequence is present on the blocker molecule. This
arbitrary sequence ensures that the length of complementary sequences
between the probe and the blocker is larger than the length of
complementary sequences between the probe and the sequences in the sample,
thus increasing the affinity of the former as compared to the latter.
It should be noted that the method is suitable for use also with several
different probes (and blockers) which are capable of hybridizing to
different regions of the nucleic acid sequences present in the sample or
several probes (and blockers) capable of hybridizing with several
different separate sequences present in the sample. The invention will now
be illustrated with reference to some non-limiting specific embodiments
described in the following with occasional reference to the annexed
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a flow chart of the method of the invention;
FIG. 2 shows the basic components of the detection ensemble;
FIGS. 3-6 show some modifications of the basic embodiment shown in FIG. 1;
FIG. 7 shows an amplification ensemble in accordance with the first
embodiment;
FIGS. 8-10 show some modifications of the amplification ensemble in
accordance with the first embodiment;
FIG. 11 shows an amplification ensemble in accordance with the second
embodiment;
FIG. 12 shows an amplification ensemble in accordance with the third
embodiment;
FIG. 13 shows a modification of the embodiment of FIG. 12;
FIG. 14 shows a detection ensemble in accordance with the fourth
embodiment;
FIG. 15 shows an amplification ensemble in accordance with the fourth
embodiment; and
FIG. 16 shows a modification of the embodiment of FIG. 15.
In the drawings, various symbols are used which in the context of the
present description have the following meanings:
Straight line (_) DNA strand
Wavy line(.about..about..about..about.) RNA strand
A, B, C, etc. . . . sequences in the coding strand of a
DNA
A', B', C', etc. . . . complementary sequences in
non-coding DNA strands
a', b', c', etc. sequences in RNA transcribed from
DNA sequences A, B, C, etc.
a, b, c, etc. . . . sequences in RNA complementary to
a', b', c', etc.
A", B" sequences in DNA which are partially
complementary to DNA sequences A
and B
TRIG triggering DNA sequence
trig triggering RNA sequence
SIG signal DNA sequence
sig signal RNA sequence
P.sup.+ functional promoter (DNA)
P- non-functional promoter (DNA)
p.sup.+ functional promoter (RNA)
p.sup.- non-functional promoter (RNA)
A-.alpha., B-.beta., A'-.alpha.', B'-.beta.', C-.gamma. complementary
sequences on the same
etc. . . . strand of nucleic acid sequence
DIS.sup.- DNA initiation sequence
In the figures, the various components are designed by three or four digit
numerals. The first digit in a case of a three digit numeral and the first
two digits in the case of a four digit numeral represent the figure number
and the last two digits represent the component number. In all figures
like components have the same component number. Thus, for example, a
component 852 in FIG. 8 has the same function as 1052 in FIG. 10, etc.
Method Overview
Reference is first made to FIG. 1 showing an overview of the method of the
present invention. Nucleic sequence 102 which is in this example a DNA
sequence, forming part of a genome of an organism in an assayed sample, is
contacted with a detection ensemble 104, comprising various DNA molecules
(as will be elaborated further below) form a first mixture 106. The
reaction mixture is subjected to conditions allowing a hybridization of
the assayed sequence 102 with corresponding sequences in the DNA molecules
of the detection ensemble (see further below).
Following hybridization and optional ligation steps, a transcription system
108 is obtained comprising a DNA heteroduplex having a double-stranded,
i.e. functional promoter and a downstream DNA sequence which can be
transcribed into an RNA molecule 110 which is referred to herein as the
triggering RNA molecule. In order to obtain the triggering RNA molecule
110, transcription reagents 113 comprising RNA polymerase and single RNA
nucleotides are added to the transcription system 108.
The triggering RNA molecule is then combined with an amplification ensemble
112 to yield a second reaction mixture 114. The amplification ensemble
comprises reagents which in the presence of the triggering RNA sequence
will give rise to the production of large quantities of an RNA molecule
116, referred to herein as the signal RNA molecule. The detection of the
presence of the signal RNA molecule can then be carried out by any number
of means known per se.
In the following, various features of the invention will be described with
reference to some specific embodiments. FIGS. 3-6 and 14 describe various
embodiments and modifications of the detection ensemble shown in FIG. 2.
FIGS. 7-13 and 15, 16 show various embodiments of the amplification
ensemble and the production of the signal RNA molecule.
Detection Ensemble
Reference is first made to FIG. 2 showing the basic features of the first
step in the performance of the method of the invention in which the
triggering RNA sequence is produced. From here on the invention will be
described with reference to the embodiments in which the assayed nucleic
acid sequence in a DNA sequence and it is to be understood that the method
is applicable also to RNA sequences, mutatis mutandis.
The detection ensemble 204 comprises a first DNA molecule 220 and a second
DNA molecule 222. The first DNA molecule 220 comprises a functional,
double-stranded promoter P.sup.+. The first DNA molecule 220 has a
single-stranded sequence A and the second DNA molecule has a
single-stranded sequence B linked to a triggering sequence TRIG which may
be single or double-stranded. The sequences A and B are complementary to
sequences A' and B', respectively, in the assayed DNA 202.
If the assayed DNA 202 is present in the sample, and appropriate conditions
for hybridization are provided, a hybrid 224 is produced. In this hybrid
the 3' end of sequence B and the 5' end of sequence A are adjacent to one
another and are optionally ligated to yield ligation product 226.
Transcription reagents 213 comprising RNA polymerase such as the T7
polymerase and RNA nucleotides and buffers are then added and as a result
a triggering RNA molecule 210 having a triggering sequence--trig linked to
sequence b' and a is produced.
Reference is now made to FIG. 3 showing a modification of the method
outlined in FIG. 3 intended to eliminate almost entirely the possibility
of obtaining a false positive result in case of an imperfect match between
the detection ensemble and the assayed DNA. The right-hand side of FIG. 3
shows the case of a perfect match between the first 320 and second 322 DNA
molecules (i.e. probes) and the assayed DNA 302; and the left-hand side of
the figure shows the case of an imperfect match, where the assayed DNA
302' comprises sequences A" and B" (the mismatch being represented
schematically by loops in sequence A and sequence B').
After hybridization between the DNA sequences (i.e. between the assayed
sequences and the probe), as described in connection with FIG. 2, the
temperature is raised to a temperature wherein there will be a total
melting of the DNA sequences in case of an imperfect match and less than
total melting, e.g. 50%, in case of a perfect match. This temperature
depends, as known, on a number of factors including the length of the DNA
sequences as well as the relative proportion of the nucleotide bases A and
T versus G and C, and has to be determined empirically in each case.
At this temperature short DNA fragments 370 and 372, being blocker nucleic
acid sequences complementary to probe sequences 320 and 322, respectively)
having sequences A' and B', respectively, are added which hybridize to the
single-stranded A and B sequences. The temperature is then lowered and a
ligase and transcription reagents are then added. In the case of an
imperfect match, the blocker molecules hybridizes with the probe molecules
released by melting in the previous step avoiding re-hybridization of the
probes to the assayed nucleic acid sequences. In such a case, only small
RNA transcripts 373 with the sequence a' will be produced whereas in the
case of a perfect match, an RNA transcript 310 having the triggering RNA
sequence--trig will be produced.
In case there is a significant difference between melting temperatures of
the above two hybrids, for example, where the hybrid in the case of any
imperfect match between the first DNA molecule and the assayed DNA has a
melting temperature T.sub.1 which is higher than melting temperature
T.sub.2 of the hybrid containing the second DNA molecule, the method may
proceed as follows: addition of first DNA molecule 320 and second DNA
molecule 322; addition of blocker molecule 370; raising temperature to
T.sub.1 ; lowering temperature to T.sub.2 ; addition of blocker molecule
372; lowering temperature to reaction temperature; addition of
transcription reagents 313.
In order to ensure that the blocker molecules 370 and 372 have an advantage
over the mismatched assayed DNA in re-hybridization with the first and
second DNA molecules (probes) when the temperature is lowered, these
blocker molecules should be in excess to the assayed DNA. In addition, it
is possible to add an extra arbitrary sequence to first molecule 320
(probe) and to add a sequence complementary to said extra sequence, to
blocker molecule 370. This extra sequence raises the affinity between the
blocker molecule and the first DNA molecule to be higher than the affinity
between the first DNA molecule and the mismatched portion of the assayed
DNA. An extra arbitrary sequence can be added in a similar manner, to
second DNA molecule and blocker molecule 372 respectively.
Reference is now made to FIG. 4 showing a modification in the method
outlined in FIGS. 2 and 3 which eliminates the production of short RNA
transcript having the sequence a', which are transcribed from the first
DNA molecule. First molecule 420 is identical to first molecule 220 in
FIG. 2. Second molecule 422' is essentially identical to second molecule
222 in FIG. 2 and is linked to a magnetic bead 418 at its 5' terminal.
Assayed DNA 402 is added to produce hybrid 424 optionally followed by
ligating to yield ligation product 426. Magnetic force is then applied.
All molecules linked to a magnetic bead, namely, free second DNA molecules
422 and ligation product 426, are drawn to the magnet 419, while molecules
unlinked to magnetic beads, namely, first DNA molecules 420 and assay DNA
molecules 402 are washed away. Transcription reagents 413 are added to the
test vessel and since the only DNA molecules containing a promoter in the
reaction mixture are ligation product 426, the only RNA transcripts which
are produced are the triggering RNA molecules 410 containing the trig
sequence. This modification enables detection of the presence of assayed
DNA by the detection of mere amplification of RNA with no need to
distinguish which type of RNA has been produced.
Reference is now made to FIG. 5 which shows another modification in the
method outlined in FIGS. 2 and 3 also intended to eliminate production of
contaminating short RNA transcripts having sequence a' transcribed from
first DNA molecules. First DNA molecule 520 contains only a
single-stranded non-functional promoter (P.sup.-). Assayed DNA 502 is
added and allowed to hybridize with first 520 and second 522 DNA molecules
and after addition of a ligase, ligation product 526 is obtained. To the
reaction mixture a blocker molecule 536 is added containing at its 5' end
a sequence which is partially complementary to part of the promoter
sequence (P.sup.- ' partial)) linked to a sequence A' complementary to
sequence A or to a part thereof. The blocker molecule 536 can hybridize
only with free first molecules 520 to yield hybrid 538 and cannot bind to
hybrid 526 since in this hybrid sequence A is already double-stranded.
Since P.sup.- ' is only partially complementary to the promoter, the
presence of mismatches in hybrid 538 makes its promoter non-functional. At
this stage, DNA molecules 540 containing a sequence P.sup.- '
complementary to the full single-stranded promoter of the first DNA
molecules are added. Molecules 540 can hybridize with hybrids 526 to give
a hybridization product 542 having a functional double-stranded promoter.
However, molecules 540 cannot hybridize with blocked hybrid 538, since the
promoter of the hybrid is already partially double-stranded. By this
modification free first molecules are blocked from forming a functional
double-stranded promoter so that when transcription reagents 513 are added
to the reaction mixture, no short RNA transcripts are produced and only
RNA triggering molecules 510 are formed. The method outlined in this
figure can be used in combination with the method of FIG. 2 in which case
blocker molecule 540 contains also sequence A" of FIG. 2 and is used to
increase the specificity of the method.
Reference is now made to FIG. 6 showing a modification of the embodiment
depicted in FIG. 1. In accordance with this embodiment, detection ensemble
604 comprises a first DNA molecule 620, a second DNA molecule 622 and a
third DNA molecule 623. These three molecules comprise single-stranded
sequences A, B, C which are complementary to corresponding sequences A'
B', C' in the assayed DNA sequence 602. Following hybridization, a
hybridization product 624 is produced which is formed from the first,
second and third DNA molecules on the one hand and the assayed DNA
sequence on the other hand. Following an optional step of ligation and a
step of addition of transcription reagents 613, a triggering RNA molecule
610 is produced in a manner similar to that described in FIG. 1.
RNA Amplification Ensemble
First Embodiment of the Invention
Reference is now made to FIG. 7 showing an RNA amplification ensemble
according to a first embodiment of the invention. According to this
embodiment, the triggering RNA molecule 710 which is a product of the
detection ensemble contains a' and b' sequences transcribed from DNA
sequence A and B. Upstream of sequence a' is sequence c' and downstream of
sequence b' is sequence .gamma.' Sequence c' and .gamma.' which are
transcribed from sequences in the first and second DNA molecules,
respectively, are arbitrary sequences complementary to each other.
Downstream of sequence .gamma. is sequence s. Sequence s is a sequence
that serves as a strong stop transcription sequence when molecule 710 is
transcribed. Sequence s is linked to a self-replicating X-RNA sequence.
The complementary sequences c'-.gamma. bring to the formation of a loop
form which functions to minimize the interference of sequences a' and b'
to the secondary structure of the X-RNA, which secondary structure is
necessary for its self-replicating activity. In the presence of RNA
transcription reagents 713, triggering RNA molecules 716 are produced,
comprising an X-RNA sequence. The X-RNA sequence serves also as the signal
molecule, the presence of which is detected by means known per se. Stop
sequence s prevents the sequences a' and b' from being transcribed from
molecule 710. Owing to its self-replicating property, large amounts of
X-RNA molecules 716 are produced within a short period of time. The
presence of large quantities of RNA will then serve as an indication for
the presence of the assayed DNA in the sample.
Reference is now made to FIG. 8 which shows a modification in the method of
the first embodiment. According to this modification, the triggering RNA
810 comprises sequences a' and b' (transcribed from the assayed DNA)
linked to sequence s which serves as a stop signal to the transcription as
described above. Sequence s is linked to an X-RNA sequence which can serve
as a template for the production of self-replicating X-RNA. This sequence
is linked to a sequence capable of forming a hairpin loop so that the
hybridized arms of this loop form a functional promoter p.sup.-, in a
manner similar to that previously reported for DNA (Kohli V. et a., Anal.
Biochem., 208, 223-227, 1993). In the presence of a transcription reagents
813, this RNA promoter enables the production of self-replicating X-RNA
816, the detection of which signifies presence of the assayed DNA in the
original sample.
FIG. 9 shows another modification of the first embodiment. According to
this modification triggering RNA molecule 910 comprises in addition to the
a', b', s and X-RNA sequences (which were described with reference to FIG.
8) the template strand of a promoter sequence p.sup.- ' Optionally, DNA
molecule 941, which comprises a sequence p.sup.- complementary to the
single-stranded RNA promoter, is added and allowed to hybridize with
molecule 910, to form DNA/RNA hybrid molecule 933 having a double-stranded
RNA/DNA promoter and an RNA sequence which serves as a template. In the
presence of transcription reagents 913, transcript 916 which is a
self-replication X-RNA sequence, is produced.
Reference is now made to FIG. 10 which shows a third modification of the
first embodiment. Triggering RNA 1010 comprises sequences, a' b', s and
X-RNA as described above. A DNA molecule 1041 is added which comprises a
double-stranded functional DNA promoter of which the non-template strand
is linked to a DNA sequence complementary to all or to part of the X-RNA
sequence of molecule 1010. The last nucleotide or several nucleotides of
the template strand of molecule 1041 is optionally an RNA nucleotide.
Molecules 1010 and 1041 are allowed to hybridize, and following ligation
of the last RNA or DNA nucleotide of molecule 1041 and the first
nucleotide of hybrid 1010, hybrid 1033 is formed. In the presence of
transcription reagents 1013 self-replicating RNA 1016 is produced.
Second Embodiment of the Invention
Reference is now made to FIG. 11 showing an RNA amplification ensemble
according to the second embodiment of the invention. According to this
embodiment, the triggering RNA molecule 1110 product of the detection
ensemble, contains sequences a' and b' linked to the triggering sequence
p.sup.- ' which is a single-stranded sequence complementary to an
essential part of the single-stranded promoter P.sup.- of a fourth DNA
molecule 1148. Fourth DNA molecule 1148 contains at the 3' end of its
template strand a single-stranded promoter sequence P.sup.- ', linked to a
double-stranded signal DNA sequence SIG, which is in turn linked to a DNA
sequence capable of being transcribed to the triggering sequence p-'
termed "promoter-sequence" in the figure. In some cases P.sup.- of
molecule 1148 may be partially double-stranded and is only single-stranded
in a part essential for the promoter's function. When RNA molecule 1110 is
added to molecule 1148, the triggering sequence p.sup.- ' of molecule 1110
hybridizes with sequence P.sup.- of molecule 1148 to form an RNA/DNA
heteroduplex 1150 having a double-stranded functional promoter P.sup.+
consisting of one DNA strand and one RNA strand or partial RNA strand.
Upon addition of transcription reagents 1113, RNA transcript 1116, which
is the signal RNA molecule comprising the RNA signal sequence "sig"and
sequence p.sup.- ', is produced. RNA transcript 1116 can in turn hybridize
with the fourth DNA molecules 1148 to produce RNA/DNA heteroduplexes 1151
which in the presence of the transcription reagents 1113 causes production
of more RNA transcripts 1116 in a self-amplifying manner. Thus, the
amounts of signal RNA molecules in the medium increases rapidly and in a
short period of time large quantities aproduced. The signal molecule can
then be detected by means known per se, either by detecting of the
presence of the specific signal sequence by merely determining the
quantity of RNA in a sample, for example, by chain light absorbance. The
presence of the signal RNA molecule indicates the existence of the assayed
DNA in the original sample.
Third Embodiment of the Invention
Reference is now being made to FIG. 12 which shows an RNA amplification
ensemble according to the third embodiment of the invention. The RNA
triggering molecule 1210, product of the detection ensemble, comprises
sequences a' and b' linked to sequences c' and d' which are complementary
to the single-stranded sequences C and D in the fifth 1252 and sixth 1254
DNA molecules, respectively. Fifth DNA molecule 1252 comprises a
double-stranded functional promoter P.sup.+ linked to a double-stranded
sequence .gamma. linked to a single-stranded sequence C. Sequence .gamma.
and sequence C are complementary to each other. Sixth DNA molecule 1254
comprises at the 3' end of the template strand a single-stranded sequence
D, linked to double-stranded sequences, C, D and a signal DNA sequence.
The two DNA molecules 1252 and 1254 are allowed to hybridize with RNA
transcript 1210 to give an RNA/DNA heteroduplex 1256. In this
hybridization product molecules 1252 and 1254 are joined together by RNA
transcript 1210. A ligase is added to ligate the adjacent ends of DNA
molecules 1252 and 1254 to yield a ligation product 1258. In the presence
of transcription reagents 1213, an RNA molecule 1216 is produced. In this
molecule sequence .gamma. and c which are complementary, form a loop. The
signal sequence sig in molecule 1216 can then be detected by means known
per se. In addition, RNA molecule 1216 can be further made to hybridize
with more fifth 1252 and sixth 1254 DNA molecules to form an RNA/DNA
heteroduplex 1260 optionally followed by ligation. In the presence of the
transcription reagents 1213 more RNA transcripts 1216 are transcribed from
hybrid 1260 which in turn cause formations of more heteroduplexes 1260,
and the reaction can continue in a self-amplifying manner.
In the presence of a transcription system 1213, fifth DNA molecules 1252,
which comprises a functional promoter produces short RNA transcripts 1262.
These short RNA transcripts however cannot interfere with the
hybridization of the fifth DNA molecules 1252 with the triggering RNA 1210
or with RNA transcript 1216, since due to the presence of complementary
sequences c and .gamma. is formed.
Reference is now being made to FIG. 13 which shows a modification of the
third embodiment of the invention. Fifth molecule 1352 comprises a
functional double-stranded promoter P.sup.+ and a short single-stranded 5'
end of sequence M. The single-stranded sequence M may be a 5' end
non-essential part of the promoter or a short linker sequence. Sixth
molecule 1354 is a DNA molecule comprising an X-DNA sequence coding for a
self-replicating X-RNA, which molecule is single-stranded in a small
region at the 3' end of the template strand having a sequence N. According
to this modification the RNA transcript 1310, the product of the detection
ensemble, comprises the a' and b' sequences attached to a single-stranded
sequence m' which is complementary to the small single-stranded region M
in fifth molecule 1352, joined to a single-stranded sequence n' which is
complementary to the single-stranded sequence N in sixth molecule 1354.
RNA transcript 1310 hybridizes with DNA molecules 1352 and 1354,and
optionally with the aid of a ligase, joins the two DNA molecules to form
an RNA/DNA heteroduplex 1364.
In the presence of transcription reagents 1313 heteroduplex 1364 is
transcribed to two products: an X-RNA transcript 1366 which is
self-replicating in the presence of a transcription system 1313; and a
transcript 1368 having as its 5' end a sequence complementary to 13 base
pairs of a promoter linked to an X-RNA sequence 1366 which is
self-replicating. RNA transcript 1368 can join additional fifth and sixth
DNA molecules to form RNA/DNA heteroduplex 1371 which heteroduplex in the
presence of a transcription system can give rise again to X-RNA molecules
1366 and transcripts 1368. The detection is then performed in a manner
similar to the above.
Fourth Embodiment of the Invention
The fourth embodiment of the invention is shown in FIGS. 14 and 15. The
first, second and third embodiments described above, make use of the same
detection ensemble and differ from one another in the amplification
ensemble. Against this, the fourth embodiment differs from the others in
both the detection as well as the amplification ensembles.
The detection ensemble in accordance with this embodiment is shown in FIG.
14. First DNA molecule 1420, which in this embodiment is completely
single-stranded, comprises at its 3' end an arbitrary sequence C, linked
to a short sequence of 1 to 5 bases termed ON and linked to a sequence A.
Second DNA molecule 1422 comprises at its 3' end a sequence B linked to an
arbitrary double-stranded sequence D-D'.
Sequence A of the first molecule is complementary to sequence A' in the 5'
end portion of the assayed DNA and sequence B of the second DNA molecule
is complementary to the sequence B' in the remaining 3' end portion of the
assayed DNA 1402.
At times, the sample contains also a sequence 1402 comprising sequences A"
and B" which are not fully complementary to sequences A and B in first and
second molecules 1420 and 1422, respectively. This may be so, for example,
in the case of genetic polymorphism. The molecules in the mixture are
allowed to hybridize, producing perfect hybridization products 1424 and
imperfect hybridization products 1424'.
In a similar manner as in the embodiment described in FIG. 3, conditions
are provided so that essentially only imperfect hybrids 1424' are melted.
A blocker molecule 1425 is added to the mixture which during cooling
hybridizes to free first DNA molecules 1420. The free first DNA molecules
include both first DNA molecules present a priori in the sample and first
DNA molecules which were freed from hybrid 1424' after melting. Molecule
1425 comprises sequences ON' and A' complementary to sequence ON and A,
respectively, in molecule 1420 and consequently hybrid 1427 is produced.
In order to ensure that all free DNA molecules will be blocked by blocker
molecules 1525, an excess of the blocker molecules is added.
To the sample are now added molecule 1429, 1431 and 1433 which together are
able to form a functional promoter with the ligation product of hybrid
1424 which is 1426 while they are not able to form a functional promoter
with hybrid 1427, thus avoiding the production of short RNA transcripts
having the sequence c on a.
Molecule 1429, termed herein "promoter molecule", comprises a
double-stranded promoter P.sup.+. One or a few of the RNA nucleotides at
the 5' end of the template strand of the promoter are optionally RNA
nucleotides. This molecule can be produced by a nucleic acid synthesizer.
The non-template strand of the promoter is linked to sequence E', C' and
ON'.
Molecule 1431 termed herein "adapter molecule" comprises a single-stranded
DNA sequence optionally having one or a few RNA nucleotides at its 3' end.
The purpose of the adapter molecule is to provide a standard sequence
having an initial RNA nucleotide which can bind to the RNA nucleotide of
the promoter at its one end and to the first DNA molecule (with the aid of
a joiner molecule) on its other end, in a case where an RNA molecule is
required on the 3' end. When an RNA molecule is not required, the adapter
molecule provides a standard sequence common to all reactions.
Alternatively, the sequence contained in the adapter molecule may be added
to each first DNA molecule when synthesized so that the need for a
separate molecule is eliminated.
Molecule 1433 termed herein "joiner molecule", it comprises at its 5' end a
part (e.g. a half) of E' and at its 3' end a part (e.g. a half) of C'.
This molecule serves to join adapter molecule 1431 and first DNA molecule
1420. In addition, hybridization with this mol renders the ON sequence
essential for hybridization of the promoter molecule 1429 to first DNA
molecule 1420. The fact that the ON sequence becomes essential avoids
binding of promoter molecule 1429 to blocked hybrid 1427 in which the ON
sequence is covered, and thus the production of short contaminating
sequences is avoided.
When the molecules are added to hybrid 1424, joiner molecule is 1433 joins
the 3' end of molecule 1420 (in the C sequence) and the 5' end of adapter
molecule 1431 (in the E sequence). After this joining, the promoter
molecule 1429 can hybridize to sequence E of the adapter molecule 1431 and
sequences C and ON in first molecule 1420. A ligase then ligates the
adjacent ends of the RNA nucleotides both in promoter molecules 1429 and
adapter molecule 1131 (when present) resulting in hybrid 1435. Adjacent
RNA nucleotides can be ligated by the T.sub.4 DNA which is known to be
able to ligate between RNA Nucleotides (Moore et al., Science, 256,
992-997, (1992). Ligation that occurs when the 3' molecule is comprised of
RNA and the 5' molecule is comprises of DNA and also efficiently ligated
by T.sub.4 DNA ligate (Nath and Hurwitz, J. Biol. Chem., 249, 3680-3688
(1974).
Promoter molecules 1429, together with the remaining components cannot bind
to blocked hybrid 1427 since in this hybrid the sequence ON is blocked,
which blockage prevents hybridization.
In the presence of transcription system 1413, RNA transcript 1410 is
produced.
The amplification ensemble is shown in FIG. 15. The ensemble compromises
promoter molecule 1529', being the same as molecule 1429 in FIG. 14, and
an opposite promoter molecule 1580 that is a promoter which is a
double-stranded promoter linked to a sequence D', complementary to
sequence d' in the transcript 1510. Transcript 1510 being identical to
1410 in FIG. 14 hybridizes to D' sequence of opposite promoter 1580 and
with the aid of a ligase an RNA/DNA hybrid 1538 is formed. In the presence
of a transcription system 1513, a new RNA transcript 1539 is transcribed.
The template of this transcript is the RNA sequence of hybrid molecule
1538. It is known that RNA can serve as a transcript for RNA production
(Leary S. L. et al, Gene, 106, 93-6 (1991). Transcript 1539 can hybridize
with promoter molecule 1529' to give hybrid 1542. The product of hybrid
1542 is again molecule 1510 which can activate opposite promoter 1580 and
so on. In this embodiment the transcription product of each hybrid 1538 or
1542 activates the reciprocal promoter in a "ping pong" manner.
The modification of the amplification ensemble of the fourth embodiment is
now shown in FIG. 16, with all identical elements to those of the original
amplification ensemble shown in FIG. 15 having the same last two digits.
RNA transcript 1610 product of the detection ensemble comprises in the
5'.fwdarw.3' direction the following sequences: one variant of the dis
sequence--a dis2' sequence which is transcribed from one variant of the
DNA initiation sequence; e', c', on', a', b', d' which are the same as
explained in FIGS. 14 and 15; and dis sequence which is another variant of
the dis sequence.
To the reaction mixture is added one type of ribozyme 1690 termed
"ribozyme1" which is able to specifically cut the dis1 sequence from the
3' end of molecule 1610 to give truncated RNA molecule 1611. RNA molecule
1611 reacts with opposite promoter 1680, which comprises a double-stranded
promoter, the coding strand of which is attached to sequence DIS1, and
optionally with the aid of a ligase, gives hybrid 1638. In the presence of
transcription reagent 1613 RNA transcript 1639 is produced, which may be
detected.
A second type of ribozyme 1591 termed "ribozyme2" which is able to cut the
dis2 sequence of transcript 1639 is used to give truncated RNA molecule
1651.
RNA molecule 1651 reacts with promoter molecule 1629, which contains a
double-stranded promoter, the coding strand of which is attached to
sequence DIS2, and optionally with the aid of a ligase gives hybrid 1642.
In the presence of transcription reagents 1613 RNA transcript 1610 is
produced again and the whole amplification cycle can restart.
Although certain presently preferred embodiments of the present invention
have been specifically described herein, it will be apparent to those
skilled in the art to which the invention pertains that variations and
modifications of the various embodiments shown and described herein may be
made without departing from the spirit and scope of the invention.
Accordingly, it is intended that the invention be limited only to the
extent required by the appended claims and the applicable rules of law.
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